The polymerase chain reaction (PCR) is utilized for the exponential amplification of one or more specific nucleic acid(s) of interest (herein "target"), particularly deoxyribonucleic acid (DNA) target(s). By utilizing primer sets which identify a particular nucleic acid sequence, a thermostable DNA polymerase (herein "polymerase"), and optimal temperature cycling, millions of copies of a nucleic acid sequence can be made from limited starting amounts. In theory, the method can amplify a single nucleic acid molecule although, in practice, more than one molecule may be required. Moreover, the PCR method generally has excellent fidelity in reproducing nucleic acid molecules identical to the target.
In the simplest terms, the polymerase faithfully produces a nucleic acid sequence that is the complement to the target. The polymerase requires that a primer bind to the target in order for the polymerase to synthesize the complement. In the case of DNA, the target and its complement form a double strand target-complement molecule. For the polymerase to produce more targets, the double strand target-complement molecule must be separated into the two single-strand molecules.
The PCR is thermally cycled to repetitively separate and form double strand target-complement molecules. Usually, the PCR is thermally cycled in a prescribed program involving three temperatures. The first temperature, approximately 94.degree. C., denatures the nucleic acid from the double strand form into the single strand form. The second temperature, typically about low 60.degree. C., anneals the primer to the single strand target. The third temperature, about 72.degree. C., allows the polymerase reaction to elongate the primers and the two strands anneal to form double strand nucleic acid. The cycle is repeated until a sufficient number of nucleic acid copies have been made. When a double stranded target is amplified, each thermal cycle amplifies the target by about 2.sup.n, where n is the number of thermal cycles completed.
PCR has various competitive side reactions that occur at relatively low temperature, generally below 60.degree. C. Competitive side reactions include, for example, mis-priming, the binding of primers to the wrong target sequence and primer dimers, the binding together of two primers. The competitive side reactions create sequences other than the target that are amplified. To counteract these reactions, a modified PCR method has been developed (herein "hot start") in which at least one key reagent, such as the polymerase or target, is withheld from the PCR mixture until the PCR mixture is heated, usually to the annealing temperature. Because the PCR cannot begin until the key reagent is added and PCR begins at the optimum temperature, the competitive side reactions are significantly reduced.
Regardless of the PCR method used, evaporation from the PCR mixture during thermal cycling must be prevented. Typically, a hydrophobic material, such as mineral oil, is layered onto the PCR reaction mixture to prevent evaporation during thermal cycling. The hydrophobic materials floats on the PCR mixture because it not miscible with and has a lower density than the PCR mixture. By completely covering the surface, the hydrophobic material seals and prevents water evaporation from the PCR mixture. A variant method is to add a small solid wax ball that melts at PCR temperatures and the melted wax covers the surface of the PCR mixture. A commercially available wax ball used for this purpose is AMPLIWAX available from PERKIN-ELMER, Norwalk, Conn., U.S.A.
A major problem with hydrophobic layering methods is the possibility of contamination. Typically, a technician loads the hydrophobic material and the technician himself is a significant potential source nucleic acid contamination. Any procedure in which a technician handles the PCR container has the potential to contaminate the sample. Precautions can be taken to reduce this risk, such as wearing gloves, but they do not guarantee contamination prevention. Methods that eliminate technician loading of the hydrophobic material are desirable because such methods significantly reduce the potential for contamination.
The current hydrophobic layering methods can also cross-contaminate target among PCRs. For example, the device used to load the hydrophobic material, such as a pipet or tweezers, can become contaminated by target and, if the same device is used repetitively, cross-contaminate other PCRs. The source of hydrophobic material, such as wax balls, can become contaminated by an amplifiable material that source will cross-contaminate other PCRs. Improper loading of the hydrophobic material can cause target to splatter out of the container and cross-contaminate other PCRs. Methods that eliminate loading the hydrophobic material are desirable because such methods significantly reduce the potential for cross-contamination by target.
Another disadvantage to current hydrophobic layering methods is that they are tedious and time-consuming. Typically, a technician carefully pipets oil, layers grease or adds a wax ball onto the surface of each PCR mixture in each individual container. This task becomes tedious to perform when a large number of PCRs are processed. More important, loading the hydrophobic material, whether manually or by automated means, takes a significant amount of time to perform when a large number of PCRs are run. A method that eliminates loading the hydrophobic material on each PCR mixture is desirable because it would significantly reduce the amount of time it takes to prepare a large number of PCRs.
Another method used to prevent evaporation is to use a microtube containing solid wax at the bottom of the tube. In this method, the PCR mixture is added on top of the solid wax at the bottom of the microtube. The wax and PCR mixture must be phase inverted by centrifugation, so that wax covers the surface of the PCR mixture when melted.
Although the current microtube containing wax method avoids many of the disadvantages of the hydrophobic layering methods, the phase inversion step is a severe limitation to this method. The phase inversion can be incomplete with PCR mixture remaining above the wax and subject to water evaporation which adversely effects the amplification reaction. Forcing the phases through each other can result in adsorption of PCR mixture in the wax. The water trapped in the wax can react violently when heated and spatter droplets of PCR mixture with obvious adverse effects on amplification. Forcing phase inversion can cause reagents in the PCR mixture to absorb onto the wax and effectively remove the reagents from the PCR. A microtube containing wax method that does not require phase inversion is desirable to reduce incomplete phase separation, splattering and reagent absorption.
Another disadvantage to the current microtube containing wax method is that the centrifugation takes time and limits sample through-put. For large numbers of samples, the centrifugation step adds a significant amount of preparation time. A microtube containing wax method that does not require a centrifugation step is desirable to reduce preparation time.
There thus exists a need for a method that prevents water evaporation during PCR without the contamination concerns and time-consuming preparation steps of current hydrophobic layering methods. Further, a method is needed that prevents water evaporation without requiring phase inversion of wax and PCR mixture. The present invention satisfies these needs and provides related advantages as well.